This invention relates generally to immunoassay detection of human carcinoma tumors by means of a labelled murine monoclonal antibody demonstrating reactivity to a specific antigen on the surface of human carcinoma cells and tissues, and more particularly, to a unique immunoassay method involving removal of a carbohydrate steric hindrance for such monoclonal antibody availability to bind the antigen for which it is specific.
The total immune system of a human includes an adaptive immune system whose function is to produce a specific reaction to an infectious disease which will enable recovery from the disease. In this function, the adaptive immune system is called upon to evolve molecules called antibodies. Antibodies are molecules produced by B lymphocytes having antigen binding portions which can recognize a determinant or antigenic site of the infectious disease or on diseased cells. A particular antibody molecule can bind only to one type of infectious disease or diseased cell or tissue. Further, each antibody molecule binds to only one of the many molecules on the disease microorganism's or diseased cell's surface. Those molecules to which antibodies bind are known as antigens.
It is known that different antibodies will bind to different antigens with each antibody being specific for a particular antigen. Any particular antigen molecule can have several different determinant sites or epitopes or may have several identical epitopes. Thus, antibodies really are specific for the epitopes or determinant antigenic sites rather than the whole antigen molecule. Thus, each antibody molecule effectively recognizes one epitope rather than the whole antigen.
In said related patent application, there is identified a murine monoclonal antibody which is specific for a unique antigenic determinant or epitope of a human carcinoma tumor cell. The antigen was called the "KC-4 antigen" and the monoclonal antibody was called "KC-4". The antigen molecule was isolated and identified as having two forms. The larger form has an approximate molecular weight of 490,000 daltons (range of 480,000 to 510,000), and occurs only in the cytoplasm of the carcinoma cells. The smaller form has an approximate molecular weight of 438,000 daltons (range of 390,000 to 450,000) and occurs both in the cytoplasm and membrane of carcinoma cells. A sample of both hybrid cell lines capable of producing monoclonal antibodies specific for this antigen are on deposit with the American Type Culture collection and are assigned the Nos. HB 8907 (IgG3) and HB 8710 (IgM).
This KC-4 monoclonal antibody is available commercially from the Coulter Immunology Division of Coulter Corporation, Hialeah, Flordia in a solid tumor marker kit under the registered trademark COULTER CLONE.RTM.. This is a carcinoma marker immunoperoxidase kit which is complete for routine use with tissues such as breast, lung, prostate, stomach and colon in the investigation of adenocarcinoma and squamous cell carcinoma. The kit can be applied to single cell suspensions (cell lines), frozen sections and paraffin embedded tissues. The immunoassay kit is available for research use to identify solid tumor markers in neoplastic tissue. The KC-4 monoclonal antibody has been available for diagnostic detection of carcinoma markers by researchers using other immunoassay techniques, including flow cytometric techniques or where the KC-4 monoclonal antibody is coated on a substrate or support member such as a microsphere for binding to KC-4 antigen.
Thus, achieving meaningful diagnostic detection of the KC-4 antigen in a pathological cell sample using routine or conventional immunoassay techniques is known. Said related patent application describes numerous immunoassay techniques for detecting the binding of the KC-4 monoclonal antibody to the KC-4 antigen on the membrane or surface of a pathological sample. In each case, the reactive binding of a stained or labelled KC-4 monoclonal antibody to the determinant site or epitope of the antigen will signal its detection. Obviously critical to such detection procedures in a pathological sample is adequate monoclonal antibody availability to bind the determinant site of the antigen for which it is specific. This presumes that the antigenic determinant is freely available without hindrance to binding with its specific monoclonal antibody at all stages of development of the human carcinoma cells, including in the very early stages of carcinoma cell development. Thus, percentage of positive carcinoma cell detection is important. Also important is being able to detect such cancer at its very early development stage where the percentage of positive antigen detection may be quite small.
I have determined that there is a carbohydrate steric hindrance to binding of the KC-4 monoclonal antibody to the smaller form of KC-4 antigen on the surface or membrane of the carcinoma cell by reason of sialic acid residue on the KC-4 antigen. The expression of the KC-4 antigen on the surface of the carcinoma cell was found to be directly related to the sialic acid on the KC-4 antigen. By removal of the sialic acid on the KC-4 antigen of the tumor cell, I have been able to enhance and/or accelerate detection, as by staining or labelling of the KC-4 antigen, which was not detectable by conventional or routine in vitro immunoassay techniques. By removing the determined carbohydrate steric hindrance on the identified KC-4 antigen, I have been able to increase the ability to detect human carcinoma tumors in a pathology sample by at least a factor of two (2). The method of the invention enhances the percentage of pathological cells detectable and even provides for earlier detection of solid tumor cells than heretofore achieved by routine immunoassay techniques.